The authors are grateful to Alessandra Moretti and Dennis Schade for their kind contribution of material. We acknowledge the great support of the iPS and EHT working group at the Department of Experimental Pharmacology and Toxicology of the UKE. The work of the authors is supported by grants from the DZHK (German Centre for Cardiovascular Research) and the German Ministry of Education and Research (BMBF), the German Research Foundation (DFG Es 88/12-1, HA 3423/5-1), British National Centre for the Replacement Refinement &amp; Reduction of Animals in Research (NC3Rs CRACK-IT grant 35911-259146), the British Heart Foundation RM/13/30157, the European Research Council (Advanced Grant IndivuHeart), the German Heart Foundation and the Freie und Hansestadt Hamburg.

<ol><li>Laverty, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+can+we+improve+our+understanding+of+cardiovascular+safety+liabilities+to+develop+safer+medicines%3F%5BTitle%5D)+AND+%22Br+J+Pharmacol%22%5BJournal%5D)">How can we improve our understanding of cardiovascular safety liabilities to develop safer medicines?</a> Br J Pharmacol. 163 (4), 675-693 (2011).</li>
<li>Guidance for Industry S7B Nonclinical Evaluation of the Potential for Delayed Ventricular Repolarization (QT Interval Prolongation) by Human Pharmaceuticals. , U.S. Department of Health and Human Services
Food and Drug Administration. Available from:
 <a target="_blank" href="http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm074963.pdf">http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm074963.pdf</a> (2005).</li>
<li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li>
<li>Burridge, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemically+defined+generation+of+human+cardiomyocytes%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Chemically defined generation of human cardiomyocytes.</a> Nat Methods. 11 (8), 855-860 (2014).</li>
<li>Kempf, H., Kropp, C., Olmer, R., Martin, U., Zweigerdt, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiac+differentiation+of+human+pluripotent+stem+cells+in+scalable+suspension+culture%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Cardiac differentiation of human pluripotent stem cells in scalable suspension culture.</a> Nat Protoc. 10 (9), 1345-1361 (2015).</li>
<li>Yang, X., Pabon, L., Murry, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+adolescence%3A+maturation+of+human+pluripotent+stem+cell-derived+cardiomyocytes%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Engineering adolescence: maturation of human pluripotent stem cell-derived cardiomyocytes.</a> Circ Res. 114 (3), 511-523 (2014).</li>
<li>Mannhardt, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+Engineered+Heart+Tissue%3A+Analysis+of+Contractile+Force%5BTitle%5D)+AND+%22Stem+Cell+Reports%22%5BJournal%5D)">Human Engineered Heart Tissue: Analysis of Contractile Force.</a> Stem Cell Reports. 7 (1), 29-42 (2016).</li>
<li>Eder, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+proarrhythmic+drugs+on+relaxation+time+and+beating+pattern+in+rat+engineered+heart+tissue%5BTitle%5D)+AND+%22Bas+Res+Cardiol%22%5BJournal%5D)">Effects of proarrhythmic drugs on relaxation time and beating pattern in rat engineered heart tissue.</a> Bas Res Cardiol. 109 (6), 436(2014).</li>
<li>Stöhr, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Contractile+abnormalities+and+altered+drug+response+in+engineered+heart+tissue+from+Mybpc3-targeted+knock-in+mice%5BTitle%5D)+AND+%22J+Mol+Cell+Cardiol%22%5BJournal%5D)">Contractile abnormalities and altered drug response in engineered heart tissue from Mybpc3-targeted knock-in mice.</a> J Mol Cell Cardiol. 63, 189-198 (2013).</li>
<li>Schaaf, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+Engineered+Heart+Tissue+as+a+Versatile+Tool+in+Basic+Research+and+Preclinical+Toxicology%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Human Engineered Heart Tissue as a Versatile Tool in Basic Research and Preclinical Toxicology.</a> PLoS One. 6 (10), e26397(2011).</li>
<li>Hansen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+drug+screening+platform+based+on+engineered+heart+tissue%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Development of a drug screening platform based on engineered heart tissue.</a> Circ Res. 107 (1), 35-44 (2010).</li>
<li>Frank, S., Zhang, M., Schöler, H. R., Greber, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Small+molecule-assisted%2C+line-independent+maintenance+of+human+pluripotent+stem+cells+in+defined+conditions%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">Small molecule-assisted, line-independent maintenance of human pluripotent stem cells in defined conditions.</a> PloS One. 7 (7), e41958(2012).</li>
<li>Lanier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Wnt+inhibition+correlates+with+human+embryonic+stem+cell+cardiomyogenesis%3A+A+structure-activity+relationship+study+based+on+inhibitors+for+the+Wnt+response%5BTitle%5D)+AND+%22J+Med+Chem%22%5BJournal%5D)">Wnt inhibition correlates with human embryonic stem cell cardiomyogenesis: A structure-activity relationship study based on inhibitors for the Wnt response.</a> J Med Chem. 55 (2), 697-708 (2012).</li>
<li>Hirt, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increased+afterload+induces+pathological+cardiac+hypertrophy%3A+a+new+in+vitro+model%5BTitle%5D)+AND+%22Bas+Res+Cardiol%22%5BJournal%5D)">Increased afterload induces pathological cardiac hypertrophy: a new in vitro model.</a> Bas Res Cardiol. 107 (6), 307-323 (2012).</li>
<li>Jacob, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+Tyrosine+Kinase+Inhibitor-Mediated+Decline+in+Contractile+Force+in+Rat+Engineered+Heart+Tissue%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Analysis of Tyrosine Kinase Inhibitor-Mediated Decline in Contractile Force in Rat Engineered Heart Tissue.</a> PLoS One. 11 (2), e0145937(2016).</li>
<li>Friedrich, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evidence+for+FHL1+as+a+novel+disease+gene+for+isolated+hypertrophic+cardiomyopathy%5BTitle%5D)+AND+%22Hum+Mol+Genet%22%5BJournal%5D)">Evidence for FHL1 as a novel disease gene for isolated hypertrophic cardiomyopathy.</a> Hum Mol Genet. 21 (14), 3237-3254 (2012).</li>
<li>Crocini, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+ANKRD1+mutations+associated+with+hypertrophic+cardiomyopathy+on+contraction+parameters+of+engineered+heart+tissue%5BTitle%5D)+AND+%22Bas+Res+Cardiol%22%5BJournal%5D)">Impact of ANKRD1 mutations associated with hypertrophic cardiomyopathy on contraction parameters of engineered heart tissue.</a> Bas Res Cardiol. 108 (3), 349(2013).</li>
<li>Fiedler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+Long+Noncoding+RNA-Based+Strategies+to+Modulate+Tissue+Vascularization%5BTitle%5D)+AND+%22J+Am+Coll+Cardiol%22%5BJournal%5D)">Development of Long Noncoding RNA-Based Strategies to Modulate Tissue Vascularization.</a> J Am Coll Cardiol. 66 (18), 2005-2015 (2015).</li>
<li>van Meer, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Small+molecule+absorption+by+PDMS+in+the+context+of+drug+response+bioassays%5BTitle%5D)+AND+%22Biochem+Biophysl+Res+Commun%22%5BJournal%5D)">Small molecule absorption by PDMS in the context of drug response bioassays.</a> Biochem Biophysl Res Commun. , (2016).</li>
<li>Godier-Furnémont, A. F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Physiologic+force-frequency+response+in+engineered+heart+muscle+by+electromechanical+stimulation%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Physiologic force-frequency response in engineered heart muscle by electromechanical stimulation.</a> Biomaterials. 60, 82-91 (2015).</li>
<li>Eng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autonomous+beating+rate+adaptation+in+human+stem+cell-derived+cardiomyocytes%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Autonomous beating rate adaptation in human stem cell-derived cardiomyocytes.</a> Nat Commun. 7, 10312(2016).</li>
<li>Huebsch, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Miniaturized+iPS-Cell-Derived+Cardiac+Muscles+for+Physiologically+Relevant+Drug+Response+Analyses%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Miniaturized iPS-Cell-Derived Cardiac Muscles for Physiologically Relevant Drug Response Analyses.</a> Sci Rep. 6 (24726), 1-12 (2016).</li>
<li>Masumoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+myocardial+regenerative+potential+of+three-dimensional+engineered+cardiac+tissues+composed+of+multiple+human+iPS+cell-derived+cardiovascular+cell+lineages%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">The myocardial regenerative potential of three-dimensional engineered cardiac tissues composed of multiple human iPS cell-derived cardiovascular cell lineages.</a> Sci Rep. 6 (29933), 1-10 (2016).</li>
<li>Ravenscroft, S. M., Pointon, A., Williams, A. W., Cross, M. J., Sidaway, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiac+Non-myocyte+Cells+Show+Enhanced+Pharmacological+Function+Suggestive+of+Contractile+Maturity+in+Stem+Cell+Derived+Cardiomyocyte+Microtissues%5BTitle%5D)+AND+%22Toxicol+Sci%22%5BJournal%5D)">Cardiac Non-myocyte Cells Show Enhanced Pharmacological Function Suggestive of Contractile Maturity in Stem Cell Derived Cardiomyocyte Microtissues.</a> Toxicol Sci. 152 (1), 99-112 (2016).</li>
<li>Zhang, J. H., Chung, T. D. Y., Oldenbutg, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Simple+Statistical+Parameter+for+Use+in+Evaluation+and+Validation+of+High+Throughput+Screening+Assays%5BTitle%5D)+AND+%22J+Biomol+Screen%22%5BJournal%5D)">A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.</a> J Biomol Screen. 4 (2), 67-73 (1999).</li>
<li>Vandenburgh, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Durg-screening+platform+based+on+the+contractility+of+tissue-engineered+muscle%5BTitle%5D)+AND+%22Muscle+Nerve%22%5BJournal%5D)">Durg-screening platform based on the contractility of tissue-engineered muscle.</a> Muscle Nerve. 37 (4), 438-447 (2008).</li>
<li>EHT Technologies GmbH. , Available from: 
 <a target="_blank" href="http://www.eht-technologies.com">http://www.eht-technologies.com</a> Forthcoming.</li>
</ol>

I.M., T.E. and A.H. are co-founders of EHT Technologies GmbH, Germany.

Engineered heart tissue offers a valuable option to the tool box of cardiovascular research. EHTs in the 24-well format have proven valuable for disease modeling8,14, drug safety screening7,8,10,11,15, or basic cardiovascular research16,17.
Potential modifications of the basic EHT protocols include studies on cellular interaction with defined mixtures of cardiomyocytes and non-cardiomyocytes18 and afterload enhancement14. Important aspects for troubleshooting include the quality of the input cell population and the quality of fibrinogen. If the initial gel formation is irregular or not compact enough to stick to PDMS posts, or if the gel is rapidly degenerated within a few days after casting due to low aprotinin concentration, EHTs generation will not be successful.
The following limitations have to be considered. (i) The throughput in 24-well format is not well suited for a primary screening setup, but is sufficient for a secondary confirmation screen. (ii) The integrated readout of force, contraction kinetics and rhythm have low specificity, which makes it difficult to link changes in force to specific molecular mechanisms. (iii) While the 3D culture format and the stretch imposed by bent PDMS posts improve morphological and functional aspects of maturation, the lack of true intercalated discs, spontaneous beating, and lower than normal adrenergic responses indicate that full cardiac maturation is not yet achieved with this protocol. (iv) PDMS has been shown to absorb different drugs to various extend19 and the actual effective concentration on the EHTs might vary. This bias might be more prominent in chronic toxicity studies15 than those investigating acute drug effects7,8 and clearly depends on the drug at hand. As a consequence, any PDMS rack exposed to drugs should be discarded after use and the potential absorption of different drugs will need to be taken into consideration when analyzing the data. (v) Given the fact that 1x106 hiPSCs are required per EHT the costs per data point are high.
The significance of this protocol compared to ex vivo heart contractility analysis is the potential to work in a human system, the lack of experimental run-down, and the standardized production of EHTs which allows setting up large studies on specific genetic backgrounds (e.g. disease modeling). In comparison to other hiPSC test systems (multi electrode array, impedance), this system analyzes contractile force as the most important physiological parameter of cardiomyocytes in a three-dimensional structure and allows cultivation under defined load. EHTs contract regularly and at stable force, frequency and rhythm over many hours and weeks, allowing long-term measurements and evaluation of chronic toxicity. EHT analysis can be performed under electrical stimulation which is important with regards to the force-frequency relationship of muscle contraction and its potential influence on drug effects. With regard to the immature status of the hiPSC-CM, electrical stimulation has the potential to push the cells towards a more mature phenotype20,21 and could be included in EHT maintenance. In addition, the EHT test system provides high content readout including all standard histological parameters as well as biochemistry (RNA, DNA, protein isolation)11. Potential future applications include aspects of drug development (target validation, safety pharmacology) and disease modeling in particular in combination with CRISPR/Cas9 mediated generation of isogenic controls.
During maintenance of EHTs and performance of concentration-response curves, it is important to carefully follow instructions for sterile cell culture procedures to avoid fungal contaminations during transfer of PDMS racks between 24-well plates. The most critical step with EHT though, is the cardiomyocyte differentiation protocol. For the EHTs to form coherently beating muscle strips, the input population has to consist of at least 60% cardiomyocytes. Fortunately, the differentiation protocols became more robust and efficient over the years and cardiomyocytes are readily commercially available by now. The role of non-myocytes in the development of hiPSC-derived engineered heart tissue is not fully understood, yet. Experiments with hiPSC-CM of high purity led to surprisingly good EHT7, but integration of other cell types into the tissue has been shown to improve tissue function and might push the phenotype towards a mature physiology22,23,24. As the tissues are generated in a 24-well-format with one million cells per tissue, this platform is still rather expensive. Scaling down to a 96-well format is aspired, but so far robustness outbalances this shortcoming. The protocol presented in this manuscript is technically very robust with little variability between the replicates. The intra-batch variability is very low for the kinetics (mean coefficient of variation = 5%), and somewhat large for the spontaneous beating frequency (mean coefficient of variation 10%) and contraction force (mean coefficient of variation 17%). The technique is very robust. Across different experimenters, the efficiency to generate beating EHTs is &#62;90% of casted hydrogels.

Cardiac side effects such as the drug-induced long QT syndrome have led to market withdrawals over the past years. Statistics indicate that about 45% of all withdrawals are due to unwanted effects on the cardiovascular system1. This drug failure after the expensive developmental process and approval is the worst-case scenario for pharmaceutical companies. Research and development departments therefore focus on detection of such unwanted cardiovascular effects early on. For economic and ethical concerns, efforts to reduce animal experiments and replace them with new in vitro screening assays are ongoing.
A set of established assays are included in the United States Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines for preclinical evaluation of proarrhythmic drug effects2. The technology of reprogramming somatic cells followed by differentiation of human induced pluripotent stem cells (hiPSC) boosted this research field3. It now offers the possibility to screen new drug candidates on human cardiomyocytes in vitro and avoids issues with inter-species differences. Recent cardiac differentiation protocols4,5 provide unlimited supply of cardiomyocytes without ethical concern. However, the measurement of contractile force, the most important and best characterized in vivo parameter of cardiomyocytes, is not well established. This is related to the relative immaturity6 of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) as compared to the adult cardiomyocyte. A possible advancement is to engineer 3-dimensional heart tissue from single cells7 (engineered heart tissue, EHT). The EHT protocol is based on embedding single murine or human cardiomyocytes8,9,10 in fibrin hydrogel between two flexible polydimethylsiloxane (PDMS) posts11 in 24-well format. Within a few days the cardiomyocytes start to contract spontaneously as single cells and start to form cellular networks. After 7-10 days, macroscopic contractions of the entire tissue are visible. During this process the extracellular matrix is remodeled, which leads to a decrease of diameter and length. The shortening of the EHT results in bending of the PDMS post even during rest, subjecting cardiomyocytes in the developing EHT to continuous load. EHTs continue to perform auxotonic muscle contractions over several weeks. Human EHTs show responses to physiological and pharmacological stimulation indicating their suitability for drug screening and disease modeling7.
In this manuscript we present a robust and easy protocol for the generation of human EHT, and the automated contractility analysis of concentration dependent changes of the contraction pattern in the presence of hERG channel inhibitors.

<table><tbody><tr><td>EHT analysis intrument</td><td>EHT Technologies GmbH</td><td>A0001</td><td>Software is included</td></tr><tr><td>EHT PDMS rack</td><td>EHT Technologies GmbH</td><td>C0001</td><td></td></tr><tr><td>EHT PTFE spacer</td><td>EHT Technologies GmbH</td><td>C0002</td><td></td></tr><tr><td>EHT electrode</td><td>EHT Technologies GmbH</td><td>P0001</td><td></td></tr><tr><td>EHT pacing adapter/cable</td><td>EHT Technologies GmbH</td><td>P0002</td><td></td></tr><tr><td>24-well-plate</td><td>Nunc</td><td>144530</td><td></td></tr><tr><td>6 well-cell culture plate</td><td>Nunc</td><td>140675</td><td></td></tr><tr><td>15 mL falcon tube, graduated&amp;nbsp;</td><td>Sarstedt</td><td>62,554,502</td><td></td></tr><tr><td>Cell scraper</td><td>Sarstedt</td><td>831,830</td><td></td></tr><tr><td>Spinner flask</td><td>Integra</td><td>182 101</td><td></td></tr><tr><td>Stirrer Variomag/ Cimarec Biosystem Direct&amp;nbsp;</td><td>Thermo scientific</td><td>70101</td><td>Adjust rotor speed to 40 rpm</td></tr><tr><td>T175 cell culture flask&amp;nbsp;</td><td>Sarstedt&amp;nbsp;</td><td>831,812,002</td><td></td></tr><tr><td>V-shaped sedimentation rack&amp;nbsp;</td><td>Custom made at UKE Hamburg</td><td>na</td><td></td></tr><tr><td>10&amp;times; DMEM</td><td>Gibco</td><td>52100</td><td></td></tr><tr><td>1-Thioglycerol&amp;nbsp;</td><td>Sigma Aldrich</td><td>M6145</td><td></td></tr><tr><td>2-Phospho-L-ascorbic acid trisodium salt</td><td>Sigma Aldrich</td><td>49752</td><td></td></tr><tr><td>Activin-A&amp;nbsp;</td><td>R&amp;amp;D systems</td><td>338-AC</td><td></td></tr><tr><td>Agarose&amp;nbsp;</td><td>Invitrogen</td><td>15510-019</td><td></td></tr><tr><td>Aprotinin</td><td>Sigma Aldrich</td><td>A1153</td><td></td></tr><tr><td>Aqua ad injectabilia</td><td>Baxter GmbH</td><td>1428</td><td></td></tr><tr><td>B27 PLUS insulin&amp;nbsp;</td><td>Gibco</td><td>17504-044</td><td></td></tr><tr><td>BMP-4</td><td>R&amp;amp;D systems</td><td>314-BP</td><td></td></tr><tr><td>Collagenase II&amp;nbsp;</td><td>Worthington</td><td>LS004176</td><td></td></tr><tr><td>DMEM</td><td>Biochrom</td><td>F0415</td><td></td></tr><tr><td>DMSO&amp;nbsp;</td><td>Sigma Aldrich</td><td>D4540</td><td></td></tr><tr><td>DNase II, type V (from bovine spleen)</td><td>Sigma&amp;nbsp;</td><td>D8764</td><td></td></tr><tr><td>Dorsomorphin&amp;nbsp;</td><td>abcam</td><td>ab120843</td><td></td></tr><tr><td>EDTA&amp;nbsp;</td><td>Roth</td><td>8043.2</td><td></td></tr><tr><td>Fetal calf serum</td><td>Gibco</td><td>10437028</td><td></td></tr><tr><td>FGF2</td><td>Miltenyi Biotec</td><td>130-104-921</td><td></td></tr><tr><td>Fibrinogen (bovine)</td><td>Sigma Aldrich</td><td>F8630</td><td></td></tr><tr><td>Geltrex&amp;nbsp;</td><td>Gibco</td><td>A1413302</td><td>For coating: 1:200 dilution</td></tr><tr><td>HBSS w/o Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;&amp;nbsp;</td><td>Gibco</td><td>14175-053</td><td></td></tr><tr><td>HEPES&amp;nbsp;</td><td>Roth</td><td>9105.4</td><td></td></tr><tr><td>Horse serum</td><td>Life technologies</td><td>26050088</td><td></td></tr><tr><td>Human serum albumin&amp;nbsp;</td><td>Biological Industries</td><td>05-720-1B</td><td></td></tr><tr><td>Insulin, human</td><td>Sigma Aldrich</td><td>I9278</td><td></td></tr><tr><td>L-Glutamin</td><td>Gibco</td><td>25030-024</td><td></td></tr><tr><td>Lipidmix&amp;nbsp;</td><td>Sigma Aldrich</td><td>L5146</td><td></td></tr><tr><td>Matrigel</td><td>BD Biosciences</td><td>354234</td><td>For EHT reconsitutionmix.</td></tr><tr><td>N-Benzyl-p-Toluenesulfonamide</td><td>TCI</td><td>B3082-25G</td><td></td></tr><tr><td>PBS w/o MgCl&lt;sub&gt;2&lt;/sub&gt;/CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Biochrom</td><td>14190</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140</td><td></td></tr><tr><td>Pluronic F-127&amp;nbsp;</td><td>Sigma Aldrich</td><td>P2443</td><td></td></tr><tr><td>Polyvinyl alcohol&amp;nbsp;</td><td>Sigma Aldrich</td><td>P8136</td><td></td></tr><tr><td>RPMI 1640&amp;nbsp;</td><td>Gibco</td><td>21875</td><td></td></tr><tr><td>Sodium selenite</td><td>Sigma Aldrich</td><td>S5261</td><td></td></tr><tr><td>TGF&amp;szlig;1</td><td>Peprotech</td><td>100-21</td><td></td></tr><tr><td>Thrombin</td><td>Sigma Aldrich</td><td>T7513</td><td></td></tr><tr><td>Transferrin&amp;nbsp;</td><td>Sigma Aldrich</td><td>T8158</td><td></td></tr><tr><td>Y-27632</td><td>Biorbyt</td><td>orb6014</td><td></td></tr><tr><td>hiPSC</td><td>Custom made at UKE hamburg</td><td>na</td><td></td></tr><tr><td>iCell cardiomyocytes kit</td><td>Cellular Dynamics International</td><td>CMC-100-010-001</td><td></td></tr><tr><td>Pluricyte cardiomyocyte kit</td><td>Pluriomics</td><td>PCK-1.5</td><td></td></tr><tr><td>Cor.4U - HiPSC cardiomyocytes kit</td><td>Axiogenesis AG</td><td>Ax-C-HC02-FR3</td><td></td></tr><tr><td>Cellartis cardiomyocytes</td><td>Takara Bio USA, Inc.</td><td>Y10075</td><td></td></tr></tbody></table>

automated contraction analysis of human engineered heart tissue for cardiac drug safety screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Cardiac Differentiation and Preparation of EHT
HiPSC were expanded on reduced growth factor basement membrane matrix, dissociated with EDTA and embryoid bodies (EBs) formed in spinner flasks overnight. After mesodermal induction for three days, cardiac differentiation was initiated with the Wnt inhibitor. After ~17 days of differentiation protocol, beating EBs were dissociated into single cells with collagenase type II (Figure 1). EHTs were prepared from freshly isolated cardiomyocytes with 1.0 x 106 cells per 100 &#181;L tissue immediately after EB dissociation (Figure 2). For preparation from frozen cells (e.g. commercial suppliers), cells were thawed by drop-wise resuspension in medium and resuspended in ECM after centrifugation. Within 48 h, spontaneous beating of single cells was observed microscopically in the EHT. Over the next days, cardiomyocytes spread out longitudinally along the force lines of the tissue, coupled and formed a coherently beating EHT. The macroscopically visible deflections of the PDMS posts were measured with the EHT analysis instrument.
Contraction Analysis
Analysis of EHT contractility was based on video-optical recording (Figure 3). Within the system, the 24-well plate with EHTs was positioned in the gas- and temperature-controlled incubator chamber with a glass roof. A camera connected to a motorized XYZ-axis was positioned above this unit, and XYZ-coordinates were defined for each EHT with a software program. Analysis of each EHT was based on figure recognition at the top and bottom ends of the tissue. An automated software algorithm analyzed the filmed EHT contractions and calculated force development based on post deflection and elasticity/geometry of the PDMS posts (Supplementary Figure 1)11. After determination of peaks according to predefined criteria, a pdf report summarized parameters of contractility for all EHTs (Supplementary Figure 2).
Stability in Long Term Measurement
Long term measurement of EHTs was performed in the continuous mode with automated repeated measurement every 20 min. Contraction analysis showed no time-dependent changes (Post test for linear trend was not significant) in contraction force, beating frequency, contraction time T1 or relaxation time T2 for up to 20 h, indicating a high level of stability (Figure 4). As with every biological sample though, the EHTs show some level of variability, as expected for biological replicates. The variation coefficients at beginning and end of long term measurement were 7/13% for force, 12/6% for frequency, 4/3% for contraction time and 4/3% for relaxation time, respectively (n = 4). This variability between biological replicates is not an artifact of figure recognition or analysis as the results for each single EHT are reproducible and have low variability (Supplementary Figure 2).
Drug Screening
Drug screening was performed in serum-free Tyrode&#39;s solution. Cumulative concentration response curves were generated with a maximum DMSO vehicle concentration of 0.1%. Baseline contraction analysis showed very little irregularity in beating pattern and very low variability between the replicates. Exposure to the hERG channel blocker E4031 resulted in a significant prolongation of the relaxation time (T2) at 10 nM and irregular beating pattern at higher concentrations (Figure 5). For frequency controlled contraction analysis, measurements were optionally performed with electrical stimulation using carbon electrodes.
<img alt="Figure 1" src="/files/ftp_upload/55461/55461fig1.jpg" /> 
Figure 1: Cardiac differentiation. (A) Human induced pluripotent stem cells. Scale bar = 100 &#181;m. (B) Embryoid bodies at the beginning of mesodermal differentiation. Scale bar = 100 &#181;m. (C) Contracting embryoid bodies at the end of cardiac differentiation. Scale bar = 100 &#181;m. (D) Human engineered heart tissue. Scale bar = 1 mm. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55461/55461fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55461/55461fig2.jpg" /> 
Figure 2: Generation of Engineered Heart Tissue (EHT). (A) PTFE spacer. (B) PDMS rack. A, (B) Scale bar = 1 cm (C) Top view on a well of a 24-well-plate with casting mold in agarose after removal of the PTFE spacer. (D) Pair of posts from the PDMS rack positioned in the casting mold. (E) Reconstitution mix pipetted into the casting mold and around the PDMS posts. (F) Freshly generated EHT at day 0, transferred to a new culture dish with medium. (G) Remodeled EHT in medium at day 15. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55461/55461fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55461/55461fig3.jpg" /> 
Figure 3: Contraction Analysis of Engineered Heart Tissue (EHT). (A) EHT analysis instrument with computer-controlled camera above the gas- and temperature-controlled incubation chamber with EHTs in 24-well-culture dish on top of a LED panel. (B) Live view of an EHT during analysis with the automated contraction analysis software. (C) Exemplary contraction pattern displaying contraction force over time measured with 100 frames per second and enlarged schematic contraction peak, displaying the analysis parameter force, contraction time (T1), relaxation time (T2), contraction velocity (CV), relaxation velocity (RV; this figure has been reprinted from7 and27). D-H: Parameters for contraction analysis. (D) Examples of different filter levels (blue filtered line) affecting the figure recognition. Left: Filter level = 0. Note that no blue filtered line is visible, but only the red original trace. Middle: Filter level = 3, indicating ideal recognition/analysis of the contraction peaks. Right: Filter level = 20. Note that the blue filtered lines are much smaller than the original traces indicating poor contraction peak analysis. (E) Example of arrhythmic EHT contractions analyzed with a too high force threshold (left) and lower force threshold (right) with all peaks recognized. (F) Examples traces of EHTs exposed to the sodium channel agonist ATX-II resulting in a prolonged relaxation time with early after contractions. Note that the fourth contraction peak on the left panel is not recognized (no green square) due to a too low minimum factor. (G) Examples of contraction peaks with (right panel) and without (right panel) pink curve (first derivative of contraction curve, contraction and relaxation velocity). (H) Summary of parameter recommendations for murine and human EHTs. Note that these parameters might need adjustment as contraction pattern changes during drug exposure (see E, F). <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55461/55461fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55461/55461fig4.jpg" /> 
Figure 4: Time Control During Long Term Measurement of Spontaneously Beating Human Engineered Heart Tissue (EHT). Time control during long term measurement of spontaneously beating human engineered heart tissue (EHT). EHTs were positioned in the EHT analysis instrument and incubated for a period of 20 h. Automated contraction analyses were performed every 20 min with not significant post-test for linear trend (n = 4; data represent mean + standard deviation). <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55461/55461fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/55461/55461fig5.jpg" /> 
Figure 5: Effect of hERG Channel Blocker E4031 on Human Engineered Heart Tissue (EHT). 
(A) Exemplary contraction pattern at baseline. (B) Exemplary contraction pattern after incubation in 300 nM E4031. (C) Average contraction peak (n = 4) of electrically stimulated EHTs at baseline (black) and after 30 min incubation in 100 nM E4031 (red). Analysis with 100 frames per second. (D) Concentration response curves for E4031 on spontaneously beating human EHTs. Note the smaller effect size in T2 prolongation in the electrically stimulated EHTs (C) vs. the spontaneously beating EHTs (D). One-way ANOVA, repeated measures with Dunnett&#39;s post-test vs. baseline; * p &#60;0.05; n= 4. Z&#39; factor25 for T2 prolongation at 300 nM E4031 is 0.75. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55461/55461fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Human</td>
			<td>Rat</td>
			<td>Mouse</td>
		</tr>
		<tr>
			<td>Cells</td>
			<td>26.4x106</td>
			<td>10.8x106</td>
			<td>18.0x106</td>
		</tr>
		<tr>
			<td>2xDMEM [&#181;L]</td>
			<td>147</td>
			<td>147</td>
			<td>147</td>
		</tr>
		<tr>
			<td>Basement membrane matrix [&#181;L]</td>
			<td>264</td>
			<td>-</td>
			<td>264</td>
		</tr>
		<tr>
			<td>Y-27632 [&#181;L]</td>
			<td>2.6</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Fibrinogen [&#181;L]</td>
			<td>66.8</td>
			<td>66.8</td>
			<td>66.8</td>
		</tr>
		<tr>
			<td>ECM [&#181;L]</td>
			<td>ad 2640</td>
			<td>ad 2640</td>
			<td>ad 2640</td>
		</tr>
	</tbody>
</table>
Table 1: Pipetting Scheme for Preparation of Reconstitution Mix for 24 Human, Rat and Mouse EHTs. Fibrinogen concentration is 5 mg/mL for all EHTs. Note that cell concentration is different for each species. For human EHTs, add basement membrane matrix (e.g. matrigel, see Materials Table) and Rho-kinase inhibitor Y-27632 to the mixture. For mouse EHTs, add basement membrane matrix. Depending on the initial concentration of the cell suspension, add additional ECM to reach a total volume of 2,640 &#181;L reconstitution mix.
<img alt="Supplementary Figure 1" src="/files/ftp_upload/55461/55461suppl1.jpg" /> 
Supplementary Figure 1: Calculation Formula for Contraction Force Based on Post Deflection26. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55461/55461suppl1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplementary Figure 2" src="/files/ftp_upload/55461/55461suppl2.jpg" /> 
Supplementary Figure 2: Exemplary Data Excerpt from the Contraction Analysis Report of the Software. 
Top: Contraction peaks over 20 s of time recorded with 100 frames/s (red: contraction force; pink: contraction velocity). Bottom: Analyzed contraction parameter showing minimum, mean, maximum value and standard deviation of the 18 analyzed contraction peaks marked with a green square (top). <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55461/55461suppl2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening at JoVE.com

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

automated-contraction-analysis-human-engineered-heart-tissue-for

Universidad San Sebastian

Research

Education

JoVE Core

JoVE Journal

Pharmacology

Bioengineering

Unit 1

Cardiovascular Drugs: Antiarrhythmic and Heart Failure Drugs

SCIENTISTS IN ACTION

12.7K Views.  University Medical Center Hamburg-Eppendorf and DZHK (German Center for Cardiovascular Research).  Hier tonen we de generering van humane gemanipuleerde hartweefsel van geïnduceerde pluripotente stamcellen (iPSC) afgeleide cardiomyocyten. We presenteren een methode om contractiekracht en voorbeeldige verandering van contractie patroon geanalyseerd door hERG kanaal remmer E-4031. Deze werkwijze vertoont een hoge mate van robuustheid en geschiktheid voor cardiale geneesmiddelscreening. 

Video: Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...

Hybrid Cell Analysis System to Assess Structural and Contractile Changes of Human iPSC-Derived Cardiomyocytes for Preclinical Cardiac Risk Evaluation

Cardiac contractility assessment is of immense importance for the development of new therapeutics and their safe transition into clinical stages. While human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold promise to serve as a human-relevant model in preclinical phases of drug discovery and safety pharmacology, their maturity is still controversial in the scientific community and under constant development. We present a hybrid contractility and impedance/extracellular field potential (EFP) technology, adding significant pro-maturation features to an industry-standard 96-well platform.
The impedance/EFP system monitors cellular functionality in real-time. Besides the beat rate of contractile cells, the electrical impedance spectroscopy readouts detect compound-induced morphological changes like cell density and integrity of the cellular monolayer. In the other component of the hybrid cell analysis system, the cells are cultured on bio-compliant membranes that mimic the mechanical environment of real heart tissue. This physiological environment supports the maturation of hiPSC-CMs in vitro, leading to more adult-like contractile responses including positive inotropic effects after treatment with isoproterenol, S-Bay K8644, or omecamtiv mecarbil. Parameters such as the amplitude of contraction force (mN/mm2) and beat duration also reveal downstream effects of compounds with influence on electrophysiological properties and calcium handling.
The hybrid system provides the ideal tool for holistic cell analysis, allowing preclinical cardiac risk assessment beyond the current perspectives of human-relevant cell-based assays.

The analysis of changes in contractile function and cellular integrity of human iPSC-derived cardiomyocytes is of immense importance for nonclinical drug development. A hybrid 96-well cell analysis system addresses both parameters in a real-time and physiological manner for reliable, human-relevant results, necessary for a safe transition into clinical stages.

Cardiac contractility assessment is of immense importance for the development of new therapeutics and their safe transition into clinical stages. While ...

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format.
The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Here, we demonstrate a non-invasive cardiac medical device contractility evaluation method using 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, plated on a flexible substrate, coupled with video-based microscopy. This tool will be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...

Preclinical Drug Testing in Scalable 3D Engineered Muscle Tissues

Accurately modeling healthy and disease conditions in vitro is vital for the development of new treatment strategies and therapeutics. For cardiac and skeletal muscle diseases, contractile force and kinetics constitute key metrics for assessing muscle function. New and improved methods for generating engineered muscle tissues (EMTs) from induced pluripotent stem cells have made in vitro disease modeling more reliable for contractile tissues; however, reproducibly fabricating tissues from suspended cell cultures and measuring their contractility is challenging. Such techniques are often plagued with high failure rates and require complex instrumentation and customized data analysis routines. A new platform and device that utilizes 3D EMTs in conjunction with a label-free, highly-parallel, and automation-friendly contractility assay circumvent many of these obstacles. The platform enables facile and reproducible fabrication of 3D EMTs using virtually any cell source. Tissue contractility is then measured via an instrument that simultaneously measures 24 tissues without the need for complex software analysis routines. The instrument can reliably measure micronewton changes in force, allowing for dose-dependent compound screening to measure the effect of a drug or therapeutic on contractile output. Engineered tissues made with this device are fully functional, generating twitch and tetanic contractions upon electrical stimulation, and can be analyzed longitudinally in culture over weeks or months. Here, we show data from cardiac muscle EMTs under acute and chronic dosing with known toxicants, including a drug (BMS-986094) that was pulled from clinical trials after patient fatalities due to unanticipated cardiotoxicity. Altered skeletal muscle function in engineered tissues in response to treatment with a myosin inhibitor is also presented. This platform enables the researcher to integrate complex, information-rich bioengineered model systems into their drug discovery workflow with minimal additional training or skills required.

This protocol provides methods for generating 3D engineered cardiac and skeletal muscle tissues and describes their use in preclinical drug screening modalities. The described methods utilize a magnetic sensing system to facilitate the simultaneous assessment of 24 tissues in parallel.

Accurately modeling healthy and disease conditions in vitro is vital for the development of new treatment strategies and therapeutics. For cardiac and ...

Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering is crucial for basic science cardiac research; in vitro human cell culture eliminates the interspecies differences of animal models, while a more tissue-like 3D environment (e.g., with extracellular matrix and heterocellular coupling) simulates in vivo conditions to a greater extent than traditional two-dimensional culture on plastic Petri dishes. However, each model system requires specialized equipment, for example, custom-designed bioreactors and functional assessment devices. Additionally, these protocols are often complicated, labor-intensive, and plagued by the failure of the small, delicate tissues.
This paper describes a process for generating a robust human engineered cardiac tissue (hECT) model system using induced pluripotent stem-cell-derived cardiomyocytes for the longitudinal measurement of tissue function. Six hECTs with linear strip geometry are cultured in parallel, with each hECT&#160;suspended from a pair of force-sensing polydimethylsiloxane (PDMS) posts attached to PDMS racks. Each post is capped with a black PDMS stable post tracker (SPoT), a new feature that improves the ease of use, throughput, tissue retention, and data quality. The shape allows for the reliable optical tracking of post deflections, yielding improved twitch force tracings with absolute active and passive tension. The cap geometry eliminates tissue failure due to hECTs slipping off the posts, and as they involve a second step after PDMS rack fabrication, the SPoTs can be added to existing PDMS post-based designs without major changes to the bioreactor fabrication process.
The system is used to demonstrate the importance of measuring hECT function at physiological temperatures and shows stable tissue function during data acquisition. In summary, we describe a state-of-the-art model system that reproduces key physiological conditions to advance the biofidelity, efficiency, and rigor of engineered cardiac tissues for in vitro applications.

Three-dimensional cardiac tissues bioengineered using stem-cell-derived cardiomyocytes have emerged as promising models for studying healthy and diseased human myocardium in vitro while recapitulating key aspects of the native cardiac niche. This manuscript describes a protocol for fabricating and analyzing high-content engineered cardiac tissues generated from human induced pluripotent stem-cell-derived cardiomyocytes.

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering ...

Contractions of Human-iPSC-derived Cardiomyocyte Syncytia Measured with a Ca-sensitive Fluorescent Dye in Temperature-controlled 384-well Plates

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac physiology and pharmacology. Various methods have been proposed to record this spontaneous activity and to evaluate drug effects, but many of these methods suffer from limited throughput and/or physiological relevance. We developed a high-throughput screening system to quantify the effects of exogenous compounds on hiPSC-CM's beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader. We describe how to prepare the cell plates and the compound plates and how to run the automated assay to achieve high sensitivity and reproducibility. We also describe how to transform and analyze the fluorescence data to provide reliable measures of drug effects on spontaneous rhythm. This assay can be used in drug discovery programs to guide chemical optimization away from, or toward, compounds affecting human cardiac function.

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells are useful models of human cardiac physiology and pharmacology. Here we present a high-throughput screening system to quantify the effects of exogenous compounds on beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader.

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac ...

Medicine

3D Human Myocardial Tissue Generation Using Melt Electrospinning Writing of Polycaprolactone Scaffolds and hiPSC-Derived Cardiac Cells

The development of functional human cardiac tissues holds significant promise for advancing applications in drug screening, disease modeling, and regenerative medicine. This protocol describes the stepwise fabrication of 3D myocardial tissues with advanced mimicry of native cardiac structure by combining melt electrospinning writing (MEW) polycaprolactone (PCL) scaffolds with fibrin hydrogels and human induced pluripotent stem cell (hiPSC)-derived cardiac cells. The process involves embedding a mixture of cardiomyocytes (hiPSC-CMs) and cardiac fibroblasts (hiPSC-CFs) within a fibrin matrix to create mini-tissues, with structural support provided by MEW-generated scaffolds. These fibrillar scaffolds are fabricated at the micro- to nanoscale, allowing for precise control over fiber architecture, which plays a key role in organizing cell distribution and alignment. Meanwhile, the fibrin matrix promotes cell viability and mimics the extracellular environment. Characterization of the generated tissues reveals well-organized sarcomeres within hiPSC-CMs, along with stable contractile activity. The tissues demonstrate consistent spontaneous beating as early as two days post-seeding, with sustained functionality over time. The combination of hiPSC-CFs with hiPSC-CMs enhances the structural integrity of the tissues while supporting long-term cell viability. This approach offers a reproducible, adaptable, and scalable method for creating biomimetic cardiac tissue models, providing a versatile platform for preclinical drug testing, mechanistic studies of cardiac disease, and potential regenerative therapies.

A reproducible method is presented to generate 3D myocardial tissues combining melt electrospinning writing (MEW) polycaprolactone (PCL) scaffolds and fibrin hydrogels with hiPSC-derived cardiomyocytes and fibroblasts. This technique offers precise control over scaffold architecture and can be applied in preclinical drug testing and cardiac disease modeling.

The development of functional human cardiac tissues holds significant promise for advancing applications in drug screening, disease modeling, and ...

Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market. Therefore, using appropriate preclinical cardiac safety assessment models is a critical step during drug development. Currently, cardiac safety assessment is still highly dependent on animal studies. However, animal models are plagued by poor translational specificity to humans due to species-specific differences, particularly in terms of cardiac electrophysiological characteristics. Thus, there is an urgent need to develop a reliable, efficient, and human-based model for preclinical cardiac safety assessment. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as an invaluable in vitro model for drug-induced cardiotoxicity screening and disease modeling. hiPSC-CMs can be obtained from individuals with diverse genetic backgrounds and various diseased conditions, making them an ideal surrogate to assess drug-induced cardiotoxicity individually. Therefore, methodologies to comprehensively investigate the functional characteristics of hiPSC-CMs need to be established. In this protocol, we detail various functional assays that can be assessed on hiPSC-CMs, including the measurement of contractility, field potential, action potential, and calcium handling. Overall, the incorporation of hiPSC-CMs into preclinical cardiac safety assessment has the potential to revolutionize drug development.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a promising in vitro model for drug-induced cardiotoxicity screening and disease modeling. Here, we detail a protocol for measuring the contractility and electrophysiology of hiPSC-CMs.

Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market. Therefore, using appropriate preclinical cardiac safety ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Samenvatting

Meer video's verkennen